In the past year, cryoelectron microscopy has enabled scientists to crank out molecular structures of tau filaments, Aβ fibrils, and huntingtin aggregates. Now researchers led by Wolfgang Baumeister, Max Planck Institute of Biochemistry, Martinsried, Germany, post another catch in the neurodegenerative arena. In the February 22 Cell, they unveil the shape of aggregates of poly-glycine-alanine, a peptide generated in amyotrophic lateral sclerosis and frontotemporal dementia. They used a version of cryoEM called cryoelectron tomography (a.k.a. cryoET) to peer at aggregates inside rat neurons. They report a dense network of twisted poly-GA ribbons studded with proteasomes, many appearing stuck in the process of chopping up unwanted protein.
- In neurons, ALS/FTD poly-Gly-Ala peptides aggregate into a dense network of twisted ribbons.
- The ribbons sequester a large fraction of the cells’ proteasomes.
- Many trapped proteasomes are frozen in a catalytic transition state.
“This is a welcome paper. There hasn’t been enough attention directed at the proteasome in the ALS field,” said Teepu Siddique, Northwestern University, Chicago. Yun-Ru (Ruby) Chen of Academia Sinica in Taipei, Taiwan, was also enthusiastic, noting the results align with her observations that poly-Gly-Ala (poly-GA) form ribbons in vitro (Chang et al., 2016). “The study will have great impact in the field,” she said. Other commentators questioned the relevance of the findings given that poly-GA’s role in amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) pathogenesis remains uncertain.
The most common genetic cause of ALS/FTD, expanded GGGGCC repeats in the C9ORF72 gene generate five different dipeptide repeat proteins within neurons. No clear correlation has yet been found between poly-GA inclusions and neurodegeneration, and poly-GR/PR peptides are considered more toxic (Freibaum and Taylor, 2017). Even so, poly-GA is most commonly found in inclusions from patient brains (Mckenzie et al., 2015; Mori et al., 2013).
To get a better look, first author Qiang Guo turned to cryoelectron tomography. While cryoET offers only nanometer resolution, not the atomic-level detail of X-ray crystallography, it works on proteins inside cells (Oikonomu and Jensen, 2017). Baumeister pioneered the approach. “This work is state-of-the-art,” said Sjors Scheres, MRC Laboratory of Molecular Biology, Cambridge, England, U.K., who solved the structure of tau fibrils from human brain with cryoEM (Jul 2017 news).
Guo transduced rat primary neurons with a construct encoding green fluorescent protein (GFP) attached to a string of 175 GA repeats. After five days, it had produced inclusions similar in size and poly-GA content to those found in C9ORF72 patient tissue (May et al., 2014). Guo flash-froze the neurons, then used an ion beam to carve out a 100–200 nm sliver containing a green aggregate visible under the light microscope. “Whole cells are too thick for the electron microscope, so we needed to thin them down,” explained co-author Rubén Fernández-Busnadiego. He placed slivers into the electron microscope and captured images at different angles by tilting the grid. Feeding the data into a computer, he obtained three-dimensional pictures (see above).
The authors were surprised by how different the aggregates looked compared with the mutant huntingtin aggregates they had recently detected using the same technique (Bäuerlein et al., 2017). “Instead of cylindrical fibrils, we saw planar, helically twisted ribbons,” said Fernández-Busnadiego. The ribbons varied in size, but were roughly 14 nm thick, up to 1 μm long, and 20 to 80 nm wide. They were densely packed, often bifurcating and merging with neighboring ribbons. The structures are similar to those described by Chen, who examined 15-repeat poly-GA peptides in vitro using transmission electron and atomic force microscopy. “We first saw fibrils forming, and then the thin ribbons,” she said.
The authors also spotted huge numbers of tiny tubes. In cross-section, they looked like 10 nm rings nestled between the poly-GA ribbons. To sharpen the view, they digitally cut out a few and aligned them to each other, generating a common structure. Reiterating this process to find the most robust structure, they realized—to their amazement—that they were staring at 26S proteasomes.
This is consistent with previous reports of proteasomes in ALS pathology (Aug 2011 news; Gorrie et al., 2014; May et al., 2014). “The results vindicate our initial identification of the causative role of the UPS pathway in ALS and ALS/FTD,” said Siddique. Still, the researchers were struck to count a 30-fold higher concentration of proteasomes in aggregates than other parts of the cell. “It’s a huge accumulation, yet the expression levels of proteasome proteins didn’t change,” said Fernández-Busnadiego, “The aggregates are sucking up the proteasomes like a sponge.”
The researchers also found 20 nm complexes corresponding to the TRiC/CCT chaperonin inside the aggregates, though these were not enriched, and saw ribosomes in the periphery of the aggregates.
Guo noticed that many of the poly-GA-associated proteasomes had bound one or two 19S regulatory particles—protein complexes that recognize, unfold, and translocate substrates into the proteasome core. The authors sorted the proteasomes on that basis, and then averaged the structural information to improve resolution, as they had done for the ribbons. They identified inactive proteasomes and others processing substrates (image at right). Interestingly, aggregates bound a large fraction of the cell’s proteasomes, and 37 percent of them were active. Normally only 20 percent of proteasomes are active at any one time. Moreover, almost a quarter of the poly-GA-bound proteasomes struck a “transient pose.” This ephemeral conformation, in which the substrate is stuck just moving into the proteolytic chamber of the proteasomes, was discovered only recently, when researchers powered proteasomes with a non-hydrolyzable form of ATP to catch them in flagrante (Wehmer et al., 2017). “They seem to be trying to degrade poly-GA, but get stuck,” said Fernández-Busnadiego. Indeed, the number of proteasomes in this state peaked near poly-GA ribbons.
What does this mean for ALS/FTD? Researchers hotly debate which repeat RNAs and proteins generated by C9ORF72 mutations are responsible for pathogenesis. Co-author Dieter Edbauer, German Center for Neurodegenerative Diseases, Munich, thinks different dipeptides play different roles in pathology, and considers poly-GA an important contributor. He told Alzforum that the group found proteasomes in 20 percent of poly-GA-positive inclusions in cortical neurons isolated from ALS patients postmortem, though that bit did not make it into the paper. “We tried poly-GR, but couldn’t see anything,” he said. CryoET sample preparation requires aggregates to be visible in the light microscope so that they can be sectioned out of the cell for subsequent EM. Poly-GA aggregates readily obliged, but poly-GR did not form visible aggregates. He also tried using induced pluripotent stem cells from patients, but these cells formed no suitable aggregates.
Several researchers wondered whether cryoET could be applied to whole tissue from people, or at least mice. Edbauer said high-pressure freezing techniques might help make this possible. “But that is going to take at least five years,” he predicted.
Can the results guide structure-based drug discovery? “The structure we have is not good enough yet. We need better resolution to find ligands,” said Edbauer. Scheres wondered if the ribbon images might be sharpened by applying the averaging trick the authors used for identifying proteasomes.—Marina Chicurel
- Tau Filaments from the Alzheimer’s Brain Revealed at Atomic Resolution
- New ALS Genes Implicate Protein Degradation, Endoplasmic Reticulum
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No Available Further Reading
- Guo Q, Lehmer C, Martínez-Sánchez A, Rudack T, Beck F, Hartmann H, Pérez-Berlanga M, Frottin F, Hipp MS, Hartl FU, Edbauer D, Baumeister W, Fernández-Busnadiego R. In Situ Structure of Neuronal C9orf72 Poly-GA Aggregates Reveals Proteasome Recruitment. Cell. 2018 Feb 8;172(4):696-705.e12. Epub 2018 Feb 1 PubMed.